| Summary: | While the potential of pluripotent cells and their role in the future of regenerative medicine has rapidly evolved since the derivation of human embryonic stem cells in 1998, ethical issues surrounding the derivation and clinical use of pluripotent cells still remain. Somatic cell reprogramming offers an ethically preferred, potentially patient specific method for deriving pluripotent cells, but this technology is based on integration of genes that have been linked to oncogenesis and therefore limit clinical usefulness. This project, started in late 2005, has explored new methods towards achieving somatic cell reprogramming with the specific goal of reprogramming human somatic cells without altering genomic DNA. Through the use of cytoplasm from pluripotent cells, total RNA from pluripotent cells, and specific mRNAs coding for known reprogramming factors, attempts to reprogram somatic cells were made, along with the goal to better understand the process of reprogramming and the associated gene expression changes that catalyse it. To gauge reactivation of the embryonic genome, an Oct4-GFP fibroblast reporter line was successfully established. A protocol for the isolation of membrane encapsulated, nuclear DNA free pluripotent cell cytoplasm, or cytoplasts, was developed. Following fusion of cytoplasts to somatic reporter cells resulted in temporary OCT4 activation, but no pluripotent cells were isolated. Subsequently, an electroporation protocol was developed and optimised to transfect total RNA and total mRNA from pluripotent cells into somatic cells, in place of cytoplasts. This method successfully showed temporary upregulation of key pluripotency genes, but not full reversion to a pluripotent state. In 2006, it was shown that only four factors (OCT4, SOX2, cMYC, and KLF4) are required for somatic cell reprogramming, but the issue of DNA manipulation remained. Synthetically produced mRNA coding for the key reprogramming factors was then made and transfected into human fibroblast cells. It was found that transfected mRNA can successfully upregulate specific genes of interest, including pluripotency factors, in a more controlled and predictable manner than DNA. Although mRNA only causes upregulation for 3-4 days, in some cases lasting changes on endogenous expression of pluripotency genes were detected, including OCT4. This work shows that mRNA transfection can be a useful tool for temporary upregulation of specific gene expression and, with further optimisation, may provide a method for catalysing somatic cell reprogramming without genetic alteration. Additionally, mRNA has potential as an important tool for differentiation, transdifferentiation, and pluripotency studies.
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